Systems for the isolation of target proteins from a suspension of host cells, e.g. microbial cells, and/or from other impurities have been developed earlier and, thus, represent prior art knowledge.
As an example, it is referred to standard protein purification procedures for the purification and isolation of soluble target proteins applying chromatographic techniques like ion-exchange or size-exclusion chromatography (Guide to Protein Purification, Academic Press Vol. 182, 1990). Other applied technologies today are liquid-liquid phase separation and ultra-filtration, among others (Guide to Protein Purification, Academic Press Vol. 182, 1990). With variations, these fundamental purification processes can be modified to purify most proteins required for scientific or industrial purposes, however, they are generally very cost intensive, complex and time consuming.
A special method to handle silk proteins in particular has been described in Huemmerich et al., Biochemistry 2004, 43, 13604-13612. In this publication a technique utilizing heat denaturation of host cell proteins following a target protein precipitation step, but lacking chromatographic purification methods, was applied successfully to purify recombinant spider silk proteins for technical applications. As silk proteins tend to self-aggregate, this method suffers from the loss of a significant fraction of the target protein, which is precipitated and, thus, unavailable for a soluble protein purification method.
Although the described methods work well for most soluble proteins, it is obvious that they bring forth severe disadvantages when handling aggregation prone proteins. Such aggregation prone proteins tend to precipitate in solution over a certain time period yielding stable, often insoluble protein aggregates. These protein aggregates can not be isolated with the described soluble protein fraction purification processes any more. Therefore, the fermentation time and/or the purification time are generally critical parameters to avoid unwanted precipitation. It is known that said protein aggregates can be solubilized using several detergents, however, it is also known that such a solubilization negatively affects the protein yield as well as the protein quality. In addition, a complete solubilization of protein aggregates over a prolonged time period is almost impossible.
Thus, there is a need to develop a purification/isolation method for aggregation prone target proteins and/or already aggregated target proteins which focuses on the separation of the aggregation prone target protein fraction and/or aggregated target protein fraction from the fraction which comprises insoluble host cell proteins and other remnants, without complete solubilization of said target proteins. Such a purification method should allow the isolation of insoluble target proteins from a suspension of host cells, e.g. microbial cells, and/or other cell remnants, in high yields and high quality. Such a purification method should also be cost effective, rapid, easy, and reproducible.
The inventors of the present invention have surprisingly found that the target proteins are insoluble and remain insoluble under certain conditions which are necessary to solubilize all or almost all other insoluble host cell proteins, host cell remnants and/or other potential fermentation-related impurities and that this allows the separation and purification of these target proteins in only a few purification steps without losing significant amounts of said target proteins due to unwanted solubilization of said target proteins or due to other cross-reactions.
Surprisingly, a purification of insoluble target proteins to preferably at least 80% purity can be achieved by separating the insoluble target proteins from the solubilized insoluble host cell parts according to the method of the present invention. Even if some insoluble host cell parts are not completely dissolved but remain in suspension, efficient separation can nevertheless be achieved as the specific density of these suspended host cell parts is lower than that of the insoluble target proteins, which allows efficient separation by centrifugation, sedimentation and/or filtration.
The method of the present invention offers several striking advantages compared to the known purification techniques. For example, the method of the present invention reduces the number of complex purification steps and is, therefore, a fast, reliable, and easy to perform purification method. In addition, said method allows the purification/isolation of the insoluble target proteins in high yield and quality. It allows a drastic cost reduction compared to conventional methods. In addition, said method is environmental friendly.
Although the method of the present invention comprises only a few method steps, it was hitherto completely unforeseeable that certain target proteins are and stay insoluble under the conditions applied. Especially when considered against the background that the applied base concentrations (˜0.1 M NaOH) are commonly used to wash and clean bioreactors as well as reaction tanks which came in contact with proteins and cells. Thus it is hardly self-evident that these conditions can also be applied to purify insoluble target proteins in large scale.